TISSUE FREEZING METHODS FOR CRYOSTAT SECTIONING Basic Tissue Freezing Methods Preparing Tissue for Freezing Then a quick overview of MHPL Cryostat sectioning Techniques: Using The Brush; Using The Anti-Roll Plate; Speaker: Donna J. Emge, HT-ASCP, MHPL Manager
Preparing Tissue For Freezing Tissue for freezing should be frozen or fixed as promptly as possible after cessation of circulation to avoid morphological distortions and damage due to: • Tissue drying artifact. • Autolysis - The destruction of tissues or cells by the action of substances, such as enzymes, that are produced within the organism. Also called self-digestion . • Putrefaction - Decomposition by microorganisms.
WHY SNAP FREEZE OR FIX AND CRYOPROTECT FOR FREEZING? Slow freezing can cause distortion of tissue due to ice crystal formation that replaces the architecture with a “Swiss Cheese” effect.
The object is to freeze so rapidly that water does not have time to form crystals and remains in a vitreous form that does not expand when solidified. Brain Skeletal Muscle Freezing artifact in sections of Brain, Muscle, and Spleen
SHORT ARTICLE ON THE SUBJECT OF WATER CRYSTAL FORMATION: “ FREEZING BIOLOGICAL SAMPLES ” Charles W. Scouten & Miles Cunningham http://www.myneurolab.com/global/Manuals/Tips%20an d%20Techniques%20Freezing%20Artifact.pdf
METHODS OF TISSUE FREEZING 1. Fresh tissue freezing – Tissue is in OCT and flash frozen fresh. 2. 4% PFA fixed, sucrose cryoprotected tissue freezing – Tissue is in OCT and may be frozen using dry ice or the flash frozen method. 3. Enzyme study tissue freezing – Often used for fresh muscle tissue. A fresh frozen method with no OCT matrix. Tissue protrudes from a Tragacanth or other support medium. MHPL Protocols for these methods are included in your workshop folder.
WHY NOT JUST USE LIQUID NITROGEN? AFTER ALL: “Liquid nitrogen is one of the coldest liquids routinely available and it does not mix with tissue.” • It Boils – this creates a vapor barrier that causes freezing in a slower, unpredictable pattern. • Tissue and OCT often cracks – due to this unpredictable freezing pattern. Review the article: “FREEZING BIOLOGICAL SAMPLES” Charles W. Scouten & Miles Cunningham http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freezing% 20Artifact.pdf
GETTING STARTED: Before you dissect the animal - organize and set-up:
Before you dissect the animal - organization and set-up: • Choose appropriate freezing method - and depending on the method prepare liquid nitrogen, isopentane, dry ice. • Label ahead of time - cryo molds, aluminum foil, specimen bags while at room temperature. • Covered Foam cooler with crushed dry ice – to temporarily hold frozen samples as you work. • Tools & other supplies - OCT, or Tragacanth, forceps, small labeled weigh boats or small labeled petri dishes.
FRESH TISSUE FREEZING Pros • Fastest of all methods. • Excellent for IHC, IF, ISH. No antigen retrieval required since no cross-linking fixative. • Often easiest to section – depending upon tissue. Cons • Poorest morphology. • Prone to freezing artifact – must be snap frozen. • ISH integrity – extreme clean techniques required or RNA will be rapidly and easily degraded.
PREPARING FRESH TISSUE FOR FREEZING (Not for enzyme study method) • Acclimate tissue to OCT - cover freshly dissected tissue for a few minutes in OCT in a labeled small petri dish or small weigh boat. • Transfer and orientate in fresh OCT in a labeled Cryomold – with just enough OCT to cover the tissue. • Avoid bubbles in the OCT – especially near the tissue. • Sectioning surface - is the bottom of the Cryomold. • Begin freezing .
Fresh Tissue Freezing Procedure: • A metal beaker is filled 2/3 with Isopentane and placed in a Dewar of Liquid Nitrogen enough to come up to about 1/3 of the metal beaker. Prepare at least 10 minutes before freezing sample. • With 12 inch forceps freeze the cryo mold prepared sample in the clear portion of isopentane – do not fully submerge . • Avoid block cracking - when there is still a small drop size of unfrozen OCT transfer sample to covered foam cooler of dry ice while continuing on to other samples.
Temporarily store frozen samples in a covered foam cooler of dry ice while continuing to freeze other samples Wrap individual samples in labeled foil, seal in a plastic bag, place in a freezer box . Store at -80° C.
4% PFA FIXED, SUCROSE CRYOPROTECTED TISSUE FREEZING Pros • Excellent morphology compared to other methods. • May use a slower freeze in crushed powder dry ice alone, slush of dry ice and 100% alcohol, or in a beaker of isopentane surrounded by dry ice - without incurring freezing artifact or block cracking. • Any of the freezing methods discussed can be used. • Good for most IHC, IF and ISH. Cons • Time consuming • Most IHC will require antigen retrieval. • Although the fixative cross-linking is protective for ISH techniques there is some RNA degradation.
PREPARING FIXED, SUCROSE CRYOPROTECTED TISSUE • 4% PFA transcardial perfuse animal. • Drop fix in 4% PFA for a few hours to O/N. • 15% sucrose in 1XPBS until tissue sinks. • 30% sucrose in 1XPBS until tissue sinks.
SUCROSE CRYOPROTECTED TISSUE FREEZING (Not for enzyme study method) • Acclimate tissue to OCT - cover freshly dissected tissue for a few minutes in OCT in a labeled small petri dish or small weigh boat. • Transfer and orientate in fresh OCT in a labeled Cryomold – with just enough OCT to cover the tissue. • Avoid bubbles in the OCT – especially near the tissue. • Sectioning surface - is the bottom of the Cryomold. • Begin freezing .
FIXED, SUCROSE CRYOPROTECTED TISSUE FREEZING • A metal beaker is ½ filled with isopentane, placed in a foam cooler or laboratory ice bucket and surround with crushed dry ice. Add a few pieces of dry ice to the isopentane and wait until boiling stops. Add more isopentane if necessary. • With 12 inch forceps freeze the cryo mold prepared sample in the isopentane – do not fully submerge. • Alternatively: Freeze cryomold prepared sample by surrounding it in finely powder crushed dry ice alone, or in a dry ice methanol or 100% Ethanol slurry. • Transfer frozen sample to a covered foam cooler of dry ice while continuing on to other samples. • Wrap all samples in labeled foil , place in a bag sealed bag, in a freezer box. • Store at -80
ENZYME STUDY TISSUE FREEZING Pros • Excellent for Enzyme histochemistry and Immunohistochemistry studies. • Best method for muscle tissue. Cons • Advanced skill needed for sectioning – no supportive OCT matrix. Anti-roll plate better than brush technique. • Time and technique skill to prepare. • Extremely susceptible to any freeze thaw – leading to loss of morphologic detail in muscle or brain tissue.
ENZYME STUDY METHOD TISSUE PREP FOR FREEZING Organize and set up:
ENZYME STUDY METHOD PREP FOR TISSUE FREEZING Prepare a small pyramid of • thick Tragacanth paste on a small piece of cork. Make a small hole in the • Tragacanth paste pyramid.
ENZYME STUDY METHOD PREP FOR TISSUE FREEZING Gently remove any surface • moisture from tissue with fresh tissue wipe. • Place 1/8 to ¼ of the tissue in a hole at top of pyramid, leaving the rest stick out above the tragacanth. • Seal edges of tragacanth to the tissue.
ENZYME STUDY TISSUE FREEZING Procedure: A metal beaker is filled 2/3 with Isopentane and placed in a Dewar of • Liquid Nitrogen enough to come up to about 1/3 to ½ of the metal beaker. Prepare at least 10 minutes before freezing sample. Hold the cork/tragacanth/sample with 12” forceps and submerge sample • side down completely into the isopentane for 10 to 20 seconds.
ENZYME STUDY TISSUE FREEZING • Transfer sample to covered foam cooler of crushed dry ice or immediately to a -80 freezer. • Rapidly wrap all samples in pre-cooled, labeled foil, and place in a pre-cooled plastic bag, in a freezer box. • Store at -80°C.
CRYOSECTIONING PREP • Remove the frozen block from the -70°C freezer and allow it to equilibrate in the cryostat chamber temperature for approximately 30 minutes. • The optimal temperature for cryostat sectioning depends on the nature of the tissue and on whether the tissues have been freshly frozen or pre-fixed with subsequent cryoprotection. • Note the reference chart for temperature setting guidelines for tissue types in your folder.
MHPL CRYOSTAT SECTIONING TECHNIQUES Using The Brush Using The Anti-Roll Plate
CRYOSTAT SECTIONING BRUSH TECHNIQUE The purpose of the brush is to grab and maneuver the section across the stage. You can buy a 1/4 inch, #2 flat, or bright brushes from an art supply store for about $3 and cut them at an angle. With this angled tip, the brush meets the tissue flat like a broom because the brush is held at an angle.
CRYOSTAT SECTIONING BRUSH TECHNIQUE Stephen R Peters M.D. Pathology Innovations, LLC http://www.pathologyinnovations.com/frozen_section_technique.htm
CRYOSTAT SECTIONING BRUSH TECHNIQUE
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